The present invention concerns a method for the detection of 5-methylcytosine in DNA. 5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. It plays an important biological role, among other things, in the regulation of transcription, in genetic imprinting and in tumorigenesis (for review: Millar et al.: Five not four: History and significance of the fifth base. In: The Epigenome, S. Beck and A. Olek (eds.), Wiley-VCH Publishers Weinheim 2003, pp. 3-20). The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. A detection of methylation is difficult, of course, since cytosine and 5-methylcytosine have the same base-pairing behavior. Many of the conventional detection methods based on hybridization thus cannot distinguish between cytosine and methylcytosine. In addition, information of methylation is completely lost in a PCR amplification.
The conventional methods for methylation analysis operate essentially according to two different principles. In the first one, methylation-specific restriction enzymes are used, and in the second one, there occurs a selective chemical conversion of unmethylated cytosines to uracil (so-called bisulfite treatment, see, e.g.: DE 101 54 317 A1; DE 100 29 915 A1). The DNA that has been pretreated enzymatically or chemically is then amplified for the most part and can be analyzed in different ways (for review: WO 02/072880 p. 1 ff). Therefore, methods which can detect methylation in a sensitive and quantitative manner are of great interest. This is true due to the important role of cytosine methylation in the emergence of cancer, particularly with respect to diagnostic applications. Of particular importance are methods which permit detection of deviant methylation patterns in body fluids, e.g., in serum. Unlike unstable RNA, DNA is often encountered in body fluids. The DNA concentration in blood in fact is increased in destructive pathological processes such as cancer disorders. A diagnosis of cancer by means of a methylation analysis of tumor DNA found in body fluids is thus possible and has in fact been described many times (see e.g.: Palmisano et al.: Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res. 2000 Nov. 1; 60 (21): 5954-8). A problem here, however, consists of the fact that in body fluids, in addition to the DNA with the methylation pattern typical of disease, there is also found a large quantity of DNA of identical sequence, but of another methylation pattern. The diagnostic methods must thus be able to detect small quantities of specifically methylated DNA against an intense background of DNA of the same sequence but of another methylation pattern.
Common methods for sensitive detection are conducted via a PCR amplification. One method is so-called methylation-sensitive PCR (“MSP”; Herman et al.: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996 Sep. 3; 93 (18): 9821-6). Here, primers are used which specifically bind only at positions of the bisulfite-treated sequence that were previously either methylated (or in the opposite approach: unmethylated). A comparable sensitive method is the so-called “heavy methyl” method. Here, a specific amplification of only the originally methylated (or unmethylated) DNA is achieved by use of methylation-specific blocker oligomers (for review: WO 02/072880). Both MSP and heavy methyl can be applied as quantifiable real-time variants. This makes possible the detection of the methylation state of a few positions directly in the course of the PCR without the need for a subsequent analysis of the products (“MethyLight”—WO 00/70090; U.S. Pat. No. 6,331,393). One embodiment of this is the “Taqman” method. This technique uses probe molecules which bear a fluorescent-dye/quencher pair. The probes hybridize in a sequence-specific manner to the amplified products and are decomposed in the course of the next amplification cycle due to the exonuclease activity of the polymerase. A detectable fluorescent signal arises due to the separation of quencher and dye (see, e.g., Eads et al.: MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 2000 Apr. 15; 28(8): E32). Another MethyLight embodiment is the so-called LightCycler method. In this case, two different probes are utilized, which hybridize to the amplified product in direct proximity to one another, and then produce a detectable signal via fluorescence-resonance energy transfer (FRET).
The applicability of this real-time method for methylation analysis, of course, is limited. This is true particularly with respect to specificity, sensitivity and reaction rate. Based on the special biological and medical importance of cytosine methylation, however, there is a great technical need for the development of higher performing methods for methylation analysis. Such a method is described in the following. Here, probes and primers are joined together so that the hybridization of the probes to the target sequence can be produced intramolecularly. The method according to the invention permits an effective and rapid detection and thus makes possible a very sensitive and very specific methylation analysis.
A method for mutation analysis that is similar to the method according to the invention has already been described under the name “Scorpion” (see, e.g.: Whitcombe et al.: Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol. 1999 August; 17(8): 804-7; Thelwell et al.: Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 2000 Oct. 1; 28(19): 3752-61; U.S. Pat. No. 6,326,145; U.S. Pat. No. 6,365,729; US 2003 0087240 A1). The Scorpion method is applicable in different embodiments. The intramolecular binding of the probe, of course, is common to all methods. In the so-called “hairpin loop” variant, the Scorpion primers bear at the 5′-end a specific probe sequence, which is present in a special hairpin loop configuration. A fluorescent dye and a quencher which are found at the ends of the probe sequence are placed in direct spatial proximity to one another by the hairpin formation. The probe and the primer sequence are joined by means of a linker, which bears a so-called PCR stopper. If, after one round of amplification, the double strand that has been formed is separated, then the probe binds intramolecularly to the elongated primer sequence of the same strand. This hybridization brings about the opening up of the hairpin, so that fluorescent dye and quencher are separated and thus a signal can be detected. The PCR stopper prevents a “read-through” of the polymerase within the PCR and thus avoids false-positive signals (see: Thelwell et al. 2000, loc. cit., particularly FIG. 1, p. 3753).
Another Scorpion variant is the so-called “duplex” method. The probe sequence is not present in a hairpin structure here, but rather forms a duplex with another oligonucleotide. Thus a fluorescent dye is bound at the 5′-end of the probe sequence, while the other oligonucleotide bears a quencher at the 3′-end. The quencher and dye are found in direct spatial proximity due to the duplex formation. If, after one round of amplification, the double strands are separated, then the probe binds intramolecularly to the elongated primer sequence of the same strand. Fluorescent dye and quencher are separated, so that a signal can be detected (Solinas et al.: Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res. 2001 Oct. 15; 29(20): E96). In addition, duplex variants are also described, in which the probes bear two dyes, and in which the signal is formed via a fluorescence-resonance energy transfer (see: Solinas et al. 2001, loc. cit., particularly p. 7 f and p. 6 FIG. 5). An advantage of the duplex method in comparison to the above-described hairpin method consists of the fact that a more intense fluorescent signal arises in the activated form due to the complete separation of quencher and dye. In addition, duplex Scorpion primers are simpler to synthesize and are less expensive than the corresponding hairpin primers (see: Solinas et al. 2001, loc. cit. p. 8 f).
Additional Scorpion variants have been described in detail in U.S. Pat. No. 6,326,145 and in US Patent Application 2003 0087240.
The Scorpion methods have several advantages when compared with conventional real-time PCR methods. This is true particularly with respect to the reaction rate. Thus the Scorpion probes hybridize intramolecularly to the target sequence and are the basis for one-molecule kinetics. In contrast, in the Taqman method, the binding of the probes takes place according to two-molecule kinetics, while in the LightCycler method, it takes place according to three-molecule kinetics. In the LightCycler method, an enzymatic degradation of the probe is also necessary, before a signal can be detected. Rapid PCR cycles, as are necessary, e.g., for high-throughput analyses, are thus possible only to a limited extent. It could be shown correspondingly that the Scorpion method is more efficient, particularly under rapid cycling conditions than the conventional real-time methodology (Thelwell et al. 2000, loc. cit.) Another advantage of the Scorpion method lies in its particular specificity. Therefore, by shortening the probe sequence, the specificity can be increased so that a single erroneous base pairing can be detected. A corresponding increase in specificity is not possible in the case of the conventional real-time variants. Shortened probes in such cases instead lead to a reduced specificity, since the probability of binding to nonspecific PCR products is increased (see: Thelwell et al. 2000, loc. cit., p. 3760).
The application of the Scorpion method to methylation analyses is described for the first time in the following. Based on the special biological and medical importance of cytosine methylation and based on the disadvantages of the known methods, the revelation of this advantageous new technology represents an important technical advance. In addition to the advantages of the Scorpion method which are already known from mutation analysis, the application of the Scorpion methodology in methylation analyses is associated with additional advantages. For example, a sensitive and specific methylation analysis is possible with the conventional PCR method only in the case of sequences which contain several co-methylated cytosine positions. In contrast, the method according to the invention in certain embodiments requires a smaller number of co-methylated positions. The Scorpion method in this case is more independent of sequence and thus has a broader field of application than the comparable known PCR method (“heavy methyl method”, see below). The use of two Scorpion primers leads to additional particular advantages. Thus, methylation and mutations can be simultaneously investigated. Also, the use of two Scorpion primers permits an internal quantification (see below).